Abstract

Mycobacterium marinum infection of poikilothermic animals, such as fish and frogs, results in chronic granulomatous diseases that bear many similarities
to mycobacterioses in mammals, including tuberculosis. This unit describes three animal models of M. marinum infection that can be used to study basic aspects of Mycobacterium‐host interactions and granuloma development, as well as trafficking of immune cells in host tissues. Protocols are included
that describe intraperitoneal infection of adult leopard frogs (Rana pipiens) and zebrafish (Danio rerio). Protocols also describe subsequent monitoring of the infection by enumeration of bacterial cfu, mean time to death, or
visual examination of infected tissue using both conventional histological stains and fluorescence microscopy of fluorescently
marked bacteria. Furthermore, protocols are included that describe the infection of embryonic zebrafish and the subsequent
analysis of the infection in real time using DIC and fluorescence microscopy.

NOTE: After inoculation, fish must be housed in a flow‐through system (see ). For most consistent results, use age‐matched fish of the same family, and acclimate fish prior to injection to ensure maximal
health. See for details.

NOTE: Standard borosilicate capillaries may also be used, but the authors find clumping of mycobacteria to be less problematic
when using aluminosilicate capillaries. If only borosilicate is available, note that the settings on a Sutter P‐2000 micropipet
puller are different for producing the same shape needle. Use: Heat=300, FIL=4, VEL=40, DEL=150, PUL=125.